Proteoform‐Resolved Interaction Studies of Plasminogen by CZE‐MS and SEC‐MS Under Near‐Native Conditions

Native mass spectrometry has become an important technique for studying proteins and protein complexes under physiologically similar conditions. However, the technique is still rarely coupled to separation techniques, as the widely used reversed-phase chromatography mostly denatures proteins. Here we present a study combining both capillary zone electrophoresis and size exclusion chromatography, each coupled online to native electrospray ionization mass spectrometry for the analysis of proteoforms of plasminogen. Plasminogen, the zymogen form of plasmin, is an abundant plasma protein with multiple proteoforms whose activation via proteolytic cleavage is tightly regulated within the fibrinolytic system and is partly modulated by post-translational modifications. Near-native CZE conditions enable the separation of proteoforms differing in phosphorylation and glycosylation, that is, almost baseline separation of proteoforms differing in phosphorylation and sialylation. The nanoflow sheath liquid interface (nanoCEasy) enables efficient ionization as well as the flexibility to ionize under native or denaturing conditions. The CZE-MS set-up can be used to study proteoform-resolved interaction directly in the capillary, as demonstrated for the activation of plasminogen by the co-injection of tissue plasminogen activator (tPA). In these proof-of-concept experiments, various truncations of plasminogen were observed with a preferred cleavage for the N-glycosylated and the phosphorylated proteoforms. SEC-MS enables partial separation of the proteoforms, particularly between glycosylated and nonglycosylated proteoforms leading to the detection of new proteoforms not described by direct infusion experiments. Overall, the combination of near-native CZE-MS and SEC-MS provides a detailed characterization of plasminogen and enables proteoforms-resolved interaction studies.